The present invention relates to tri- and tetra-valent monospecific antigen-binding proteins and to methods for their production as well as to tri- and tetra-valent ligands for their construction. The invention relates in particular, but not exclusively, to the use of recombinant DNA technology to produce such tri- and tetra-valent monospecific antigen-binding proteins.
There has been much interest in recent years in antibodies and their fragments. It is well known that complete antibody molecules are made up of heavy chain and light chain heterodimers. For instance an IgG molecule comprises four polypeptide chains, two heavy-light chain heterodimers. Each light chain consists of two domains, the N-terminal domain being known as the variable or VL domain and the C-terminal domain being known as the constant or CL domain. Each heavy chain consists of four or five domains, depending on the class of the antibody. The N-terminal domain is known as the variable or VH domain. This is attached at its c-terminal end to the N-terminal end of the next domain, which is known as the first constant or CH1 domain. The next part of each heavy chain is known as the hinge region and this is then followed by the second, third and, in some cases, fourth constant or CH2, CH3 and CH4 domains respectively.
In an assembled antibody, the VL and VH domains associate together to form an antigen binding site. Also, the CL and CH1 domains associate together to keep one heavy chain associated with one light chain. Two heavy-light chain heterodimers associate together partly by interaction of the CH2, CH3 and, if present, CH4 domains of the two heavy chains and partly because of interaction between the hinge regions on the two heavy chains.
Each heavy chain hinge region includes at least one, and often several, cysteine residues. In the assembled antibody, the hinge regions of the heavy chains are aligned so that inter-chain disulphide bonds can be formed between the cysteine residues in the hinge regions, covalently bonding the two heavy-light chain heterodimers together. Thus, fully assembled antibodies are at least bivalent in that they have at least two antigen binding sites.
It has been known for some long time that if the disulphide bonds in an antibody""s hinge region are broken by mild reduction, it is possible to produce a monovalent antibody comprising a single heavy-light chain heterodimer.
It has also been known for some long time that treatment of antibodies with certain proteolytic enzymes leads to the production of various antibody fragments. For instance, if an antibody is cleaved close to the N-terminal side of each hinge region, two antigen binding fragments (Fab) and one constant region fragment (Fc) are produced. Each Fab fragment comprises the light chain associated with a truncated heavy chain comprising only the VH and CH1 domains. The Fc portion comprises the remaining domains of the heavy chains held together by the hinge region. Alternatively, the antibody may be cleaved close to the C-terminal side of the hinge. This produces a fragment known as the F(abxe2x80x2)2 fragment. This essentially comprises two Fab fragments but with the CH1 domains still attached to the hinge regions. Thus, the F(abxe2x80x2)2 fragment is a bivalent fragment having the two antigen binding sites linked together by the hinge region. The F(abxe2x80x2)2 fragment can be cleaved by reduction to produce a monovalent Fabxe2x80x2 fragment. This can be regarded as being a Fab fragment having on it a hinge region.
It has also proved to be possible, by careful control of digestion conditions, to cleave an antibody between the VL and CL and between the VH and CH1 domains. This gives rise to two fragments known as Fv fragments. Each FV fragment comprises a VL and a VH domain associated with one another. Each Fv fragment is monovalent for antigen binding.
Studies of the amino acid sequence of individual variable domains has shown that there are three areas in each variable domain where the sequence varies considerably. These areas have been termed hypervariable regions or complementarity determining regions (CDRs). The location of these CDRs has been published [Kabat, E. A. et al., in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA, 1987 and Wu, T. T. and Kabat, E. A., J. Exp. Med., 132, 211-250, 1970].
Structural studies on crystallised Fv fragments and molecular modelling studies have shown that each variable domain consists of three loop regions supported on xcex2-pleated sheet framework regions. In the case of hapten antigen binding the loop regions appear to form a pocket for receiving the antigen.
There is considerable overlap between the CDRs, as determined by sequence analysis, and the loop regions, as determined by structural analysis. However, it is generally accepted that the CDRs, possibly in combination with some extra residues present in the loop region, are primarily involved in determining the antigen binding specificity of the antibody.
In more recent years, there has been much interest in producing antibodies or their fragments by use of recombinant DNA technology. The patent literature is replete with disclosures in this area. Recombinant DNA technology has been used not only to reproduce natural antibodies but also to produce novel antibodies. For instance, it is now possible to produce chimeric antibodies, wherein the variable domains from one species are linked to constant domains from another species.
It is also possible to produce modified antibodies, in which the residues in the CDRs and, if necessary, a number of other residues in the variable domains have been changed so that a different antigen can be bound. This is a useful procedure in that it allows a specificity from, for instance, a mouse monoclonal antibody (MAb) to be created in a human antibody without altering the essentially human nature of the antibody. This has advantages where it is desired to use the antibody in vivo. A further discussion is given in W0-A-91/09967.
WO-A-90/09195 and WO-A-90/09196 relate to cross-linked antibodies and processes for their preparation. Cross linked antibody conjugates are described which have at least one non-disulphide (Sxe2x80x94S) interchain bridge optionally containing a reporter or effector molecule. The bridge may be the residue of a homo- or hetero-functional cross-linking reagent and is located away from the antigen binding domains of the antibody. The antibody conjugates have an enhanced binding capacity, in vivo have good blood clearance and, in the presence of a tumour, high tumour: blood and tumour : bone ratios. The conjugates are of use in the diagnosis and therapy of tumours.
They may be prepared by reaction of a cross-linking reagent with an antibody or a fragment thereof. The cross-linking reagent may react either with thiol groups on the antibody molecules or with the side chains of amino acid residues such as glutamic acid, aspartic acid, lysine or tyrosine residues.
However, we have found that while cross linked antibodies as described in WO-A-90/09195 and WO-A-90/09196 have improved properties over natural immunoglobulins and in particular exhibit highly successful binding to tumour cells and good clearance from the blood, they are subject to high uptake by the kidneys and are retained in this tissue. This creates a toxicity problem, particularly when the antibody is radiolabelled for use in therapy and radioimaging. What is required is therefore an antibody molecule which retains the superior binding and clearance properties of cross-linked antibodies but which is not taken up or retained by kidney tissue and thus avoids kidney toxicity problems.
WO-A-91/03493 relates to bi- or tri-valent multispecific Fab conjugates. The conjugates which are described comprise three or four Fabxe2x80x2 antibody fragments linked together using orthophenylenedimaleimide bridging structures. The disclosed trimeric conjugates comprise either two Fabxe2x80x2 fragments of a first specificity and one Fabxe2x80x2 fragment of a second specificity or three different Fabxe2x80x2 fragments each of different specificities. Thus, the trimeric conjugates are either bi-or tri-specific. In a similar fashion, the disclosed tetrameric conjugates are at least bispecific and may be tri- or tetra-specific.
It is reported in WO-A-91/03943 that, in certain circumstances, a population of T lymphocytes can be induced to kill target cells, such as tumour cells, by treatment with a bispecific dimeric conjugate, wherein one specificity is directed at a specific antigenic structure on the T-lymphocyte population and the other specificity is directed at an antigen on the target cells. This effect is referred to as redirect cellular cytotoxicity (RCC).
The invention disclosed in WO-A-91/03493 is based on the assertion that RCC can be significantly improved by use of trimeric or tetrameric multispecific conjugates. Use of such conjugates also allows the range of T lymphocyte antigens which can be specific to be increased. It is thus essential to the invention claimed in WO-A-91/03493 that the tri- or tetra-meric conjugates should be at least bispecific.
A more detailed discussion of the invention disclosed in WO-A-91/03493 is found in Tutt, A. et al., Eur. J. Imunol., 21, 1351-1358, 1991, which confirms that it is essential, in order to enhance RCC, to use tri- or tetra-meric conjugates which are at least bispecific. However, it should be noted that nowhere in WO-A-91/03493 or Tutt et al., supra, are the clearance properties of tri- or tetra-meric Fab conjugates discussed.
It has been suggested, in our copending International Patent Specification No. W091/19739, that multivalent antigen-binding Fv fragments will be of use in imaging or treating tumours in vivo.
A further requirement for multivalent antigen binding proteins such as those discussed above is for a cross linking molecule capable of cross linking antibody fragments together. In addition to its cross linking function, such a cross linking molecule can advantageously provide for the introduction of effector or reporter molecules to the antibody conjugate.
A number of cross-linking molecules have been described.
For example, European Patent specification No. 0446071 (Hybritech Incorporated) discloses the production of tri-functional cross linkers for use in the production of bi-specific trimeric antibody-like molecules. The application of such tris-maleimide compounds to the production of bi- or tri-specific trivalent antibody-like compounds is disclosed in European Patent Application 0453082 (Hybritech Incorporated). The clearance properties of the antibody conjugates disclosed are not referred to. A distinct drawback of the disclosed linkers is that it is difficult to attach a functional group such as a radioisotope thereto.
In particular a macrocycle cross-linking group is not easily incorporated into such linkers.
The present invention is based on the discovery that tri- and tetra-valent monospecific Fab-like proteins are particularly suitable for anti-cancer therapy. These proteins demonstrate the superior binding and clearance properties of cross-linked antibodies but are not taken up and/or retained by non-tumour tissues, including kidney tissue. In addition, the present invention provides novel linker molecules which greatly facilitate the attachment of reporter or effector groups to tri- or tetra-valent Fab-like proteins.
Therefore, according to the present invention, there is provided a tri- or tetra-valent monospecific antigen-binding protein comprising three or four Fab fragments bound to each other by a connecting structure, which protein is not a natural immunoglobulin.
The multivalent antigen-binding proteins of the invention are referred to herein as TFM (tri-Fab) and QFM (tetra-Fab). It will be understood that the expression xe2x80x9cFabxe2x80x9d is used herein to include optionally modified Fab and Fabxe2x80x2 antibody fragments derived from natural antibodies or synthesised, either chemically or by recombinant DNA technology. By xe2x80x9coptionally modifiedxe2x80x9d is meant that the Fab or Fabxe2x80x2 fragment may contain a number of insertions, deletions or changes in the amino acid sequence, as long as the binding ability of the fragment is not adversely affected.
Preferably, in compounds according to the invention the Fab fragments are bound together covalently by the use of a single linker molecule.
Surprisingly, it has been observed that TFM and QFM have markedly superior characteristics to whole antibodies, Fab, F(abxe2x80x2 )2 and monospecific cross-linked derivatives of these fragments. While Fab, F(abxe2x80x2)2 and their cross-linked counterparts are relatively specific for tumour cells when used in vivo, TFM and QFM show a greatly increased avidity compared therewith. At the same time, they are eliminated from the blood much more efficiently than whole antibodies. Furthermore, in contrast to previously described monospecific cross-linked Fab and F(abxe2x80x2)2 fragments, TFM and QFM do not accumulate in the kidney. This gives rise to a decrease in undesirable side-effects, particularly where the antibody molecule is conjugated to a toxin or a radioisotope for anticancer therapy.
Preferably, the multivalent Fab-like proteins of the invention are specific for a tumour-associated antigen. Advantageously, therefore, at least the CDRs of the Fab fragments are derived from a tumour-specific monoclonal antibody (MAb). Alternatively, the CDRs may be synthetic.
It will be appreciated that any tumour-specific antigen May be targetted by the Fab-like proteins of the present invention.
The TFM or QFM compounds of the invention may be labelled by one or more reporter or effector groups, for example the types described below. The label may be incorporated on the Fab portion of the TFM or QFM molecule, and/or on the connecting structure linking the Fab portions to each other. Where the Fab portion itself is labelled, the label will generally be located such that it does not interfere with the binding site of the fragment. Methods of labelling antibodies with a reporter or effector group are well known, and are described in our published patent specifications EP 238196, EP 384624, EP 385601, WO88/05433, WO89/01475, WO89/01476 and WO90/01475. Where it is desired to include a reporter or effector group in the connecting structure, this may be achieved by reaction of the reporter. or effector group with a reactive functional group present in the connecting structure, for example in analogous fashion to that used for the labelling of the Fab fragment, or the reporter or effector group may be advantageously built in to the connecting structure, for example as described below.
Preferably, in the TFM and QFM compounds of the invention, the Fab monomers are cross-linked together by a cross-linker. The cross-linker may be any chemical capable of linking the Fab fragments together. Preferably, however, the cross-linker is a specifically designed chemical compound such as the maleimide compounds described in EP-A-0446071 and EP-A-0453082, although it will be understood that any structure having three or four functional groups reactive with any reactive amino acid found on an antibody chain may be used.
In one preference the connecting structure in the compounds of the invention is a polylysine linker. According to a second aspect of the invention, therefore, we provide a cross-linking agent of formula (1);
R1CH(R2)NHCOR3xe2x80x83xe2x80x83(1)
wherein R1 is a carboxyl (xe2x80x94CO2H) or esterified carboxyl (xe2x80x94CO2R) group or a group xe2x80x94COA where A is an effector or reporter molecule attached to the xe2x80x94CO group either directly or via a spacer group to form a carbon-carbon, or carbon-hetero atom linkage; R2 and R3, which may be the same or different, is each an optionally substituted straight or branched alkylene, alkenylene or alkynylene chain [optionally interrupted by one or more xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94 atoms, or xe2x80x94N(R4) (where R4is a hydrogen atom or a C1-6 alkyl group), xe2x80x94N(R4)COxe2x80x94, xe2x80x94CON(R4)xe2x80x94, C5-8 cycloalkylene, C6-12 arylene or C5-10 heteroarylene groups) containing one or more reactive functional groups such that the total number of reactive functional groups in R2 and R3 together is three or more.
In the compounds of formula (1), the term xe2x80x9ceffector groupxe2x80x9d is to be understood to mean any group capable of eliciting a change in, or a response from, a biological system and which also confers this property to the compound of formula (1). The term xe2x80x9creporter groupxe2x80x9d is to be understood to mean any group which is detectable by analytical means in vitro and/or in vivo and which confers this property to the compound of formula (1).
Effector. groups include, for example, any physiologically active substance, antibacterial, antiviral or antifungal compound. Particular physiologically active substances include antineoplastic agents, toxins (such as enzymatically active toxins of bacterial or plant origin and fragments thereof e.g. ricin and fragments thereof), enzymes, anti-flammatory compounds and substances active as cardiovascular, e.g. fibrinolytic, and central nervous system, agents.
Particular antineoplastic agents include cytotoxic and cytostatic agents, for example alkylating agents, such as nitrogen mustards (e.g. chlorambucil, melphalan, mechlorethamine, cyclophosphamide, or uracil mustard) and derivatives thereof, triethylenephosphoramide, triethylenethiophosphoramide, busulphan, or cisplatin; antimetabolites, such as methotrexate, fluorouracil, floxuridine, cytarabine, mercaptopurine, thioguanine, fluoroacetic acid or fluorocitric acid, antibiotics, such as bleomycins (e.g. bleomycin sulphate), doxorubicin, daunorubicin, mitomycins (e.g. mitomycin C), actinomycins (e.g. dactinomycin) plicamycin, calichaemicin and derivatives thereof, or esperamicin and derivatives thereof; mitotic inhibitors, such as etoposide, vincristine or vinblastine and derivatives thereof; alkaloids, such as ellipticine; polyols such as taxicin-I or taxicin-II; hormones, such as androgens (e.g. dromostanolone or testolactone), progestins (e.g. megestrol acetate or medroxyprogesterone acetate), estrogens (e.g. dimethylstilbestrol diphosphate, polyestradiol phosphate or estramustine phosphate) or antiestrogens (e.g. tamoxifen); anthraquinones, such as mitoxantrone, ureas, such as hydroxyurea; hydrazines, such as procarbazine; or imidazoles, such as dacarbazine.
Particularly useful effector groups are calichaemicin and derivatives thereof (see for example South African Patent Specifications Nos. 85/8794, 88/8127 and 90/2839).
Suitable reporter groups include chelated metals, fluorescent compounds or compounds which may be detected by NMR or ESR spectroscopy.
Chelated metals include chelates of di- or tripositive metals having a coordination number from 2 to 8 inclusive. Particular examples of such metals include technetium (Tc), rhenium (Re), cobalt (Co), copper (Cu), gold (Au), silver (Ag), lead (Pb) bismuth (Bi), indium (In), gallium (Ga), yttrium (Y), terbium (Tb), gadolinium (Gd), and scandium (Sc). In general the metal is preferably a radionuclide. Particular radionuclides include 99mTc, 186Re, 188Re, 58Co, 60l Co, 67Cu, 195Au, 199Au, 110Ag, 203Pb, 206Bi, 207Bi, 111In, 67Ga, 68Ga, 88y, 90Y, 160Tb, 153Gd and 47Sc.
The chelated metal may be for example one of the above types of metal chelated with any suitable polydentate chelating agent, for example cyclic polyamines, polyethers, (e.g. crown ethers and derivatives thereof); polyamides; porphyrins; and carbocyclic derivatives.
In general, the type of chelating agent will depend on the metal in use. One particularly useful group of chelating agents in conjugates according to the invention, however, are acyclic and cyclic polyamines, especially polyaminocarboxylic acids, for example diethylenetriaminepentaacetic acid and derivatives thereof, and macrocyclic amines, e.g. cyclic tri-aza and tetra-aza derivatives; and polyamides, especially desferrioxamine and derivatives thereof.
Examples of particular macrocyclic amines include compounds of formula (2): 
(wherein L is a substituent containing a reactive group, B is a C2-4 alkylene chain interrupted by one or two optionally substituted nitrogen atoms; W1 and W2, which may be the same or different, is each an optionally substituted nitrogen atom; p is zero or an integer 1 and q is zero or an integer 1 or 2 with the proviso that when p is zero, q is an integer 1 or 2). It will be appreciated that the group L provides an attachment point for the macrocycle to the rest of the compound of formula (1). Typical groups include for example amine (xe2x80x94NH2) containing groups. Preferred amines of formula (2) include tri-aza derivatives of formula (3): 
wherein W1 and W2 which may be the same or different is each a group xe2x80x94[CH2)rR1]xe2x80x94 (where r is zero or an integer 1 to 6 and R1 is an alkyl, alkoxyalkyl, xe2x80x94CO2H, xe2x80x94SO3H, xe2x80x94PO3H2 or aryl group) and B is a group xe2x80x94CH2(CH2)8N(R)(CH2)tCH2xe2x80x94 (where s and t, which may be the same or different is each zero or an integer 1, 2 or 3; and R represents xe2x80x94(CH2)rR1 where r and R1 are as just described)]; and tetra-aza derivatives of formula (4); 
[wherein W1 and W2 which may be the same or different is each a group xe2x80x94N[(CH2)rR1]xe2x80x94 (as just defined) and B is a group xe2x80x94CH2(CH2)sN(R)CH2(CH2)dN(R)(CH2)tCH2 (where d is zero or an integer 1, 2 or 3 and s, t and R are as just defined].
A particularly useful amine of formula (3) is the compound of formula (5) 
A particularly useful amine of formula (4) is the compound of formula (6): 
Preferred chelated metal s in conjugates according to the invention include indium chelated by a compound of formula (3), particularly the compound of formula (5); or yttrium chelated by a compound of formula (4), particularly the compound of formula (6). 111In and 90Y are particularly preferred.
The effector or reporter group may in general be attached to the remainder of the compound of formula (1) via any suitable carbon atom or heteroatom, e.g. nitrogen, oxygen, sulphur or phosphorous atom, present in it, either directly to form a compound Axe2x80x94COCH(R2)NHCOR3 or indirectly to form a compound A-Sp-COCH(R2)NHCOR3 where Sp is a spacer group attached independently to A and to xe2x80x94COxe2x80x94 group through a carbon-carbon or carbon-heteroatom linkage as just described. Suitable spacer groups include acylic or cyclic aliphatic or aromatic residues in particular alkylene [e.g. ethylene, propylene, butylene], alkoxyalklene [e.g. methoxymethylene, ethoxymethylene, ethoxyethylene], arylene [e.g. phenylene] aralkylene [e.g. phenalkylene such as phenethylene] or cycloalkylalkylene [e.g. cyclohexylmethylene]groups.
The linkage between A and the group xe2x80x94COxe2x80x94 or A and the spacer group may if desired be chosen so as to be cleavable, such as by proteolytic enzymes, for example as described in European Patent Specification No. 175617.
Esterified carboxyl (xe2x80x94CO2R) groups represented by R1 in compounds of formula (1) include those groups wherein R is an organic group, for example an acyclic aliphatic group, or an aromatic or heteroaromatic group.
Thus R may be an optionally substituted straight or branched C1-2 alkyl, (e.g. methyl, ethyl, n-propyl, i-propyl, s-propyl, n-butyl, i-butyl, s-butyl, t-butyl) , C2-20 alkenyl, or C2-20 alkynyl group optionally interrupted by one or more xe2x80x94Oxe2x80x94or xe2x80x94Sxe2x80x94 atoms; or a C5-8 cycloalkyl (e.g. cyclopentyl or cyclohexyl), C5-8 cycloalkyl C1-6 alkyl (e.g.cyclopentylmethyl, cyclohexylmethyl), C6-12 aryl (e.g. optionally substituted phenyl or naphthyl) C6-12 or C1-6 alkyl (e.g. optionally substituted benzyl, phenethyl, or naphthylmethyl), C5-10 heteroaryl (e.g. furanyl, pyridyl, thienyl) or C5-10 heteroaryl C-1-6 alkyl (e.g. furanylmethyl, pyridylmethyl, or thienylmethyl) group.
The reactive functional group in compounds of formula (1) may in general be any group capable of reacting with a thiol, amino, carboxyl, hydroxyl, aldehyde, aromatic or heteroaromatic group. Aromatic groups include, for example, phenolic groups. Heteroaromatic groups include, for example, imidazolyl groups.
Thus, the reactive functional group may be, for example, a halogen atom, for example a chlorine, bromine or iodine atom, or a group selected from xe2x80x94SH, xe2x80x94Sxe2x80x94S-Het (where Het is an optionally substituted heterocyclic group, e.g. an optionally substituted pyridyl group), xe2x80x94NH2, hydrazine (NHHH2) or a derivative thereof, [for example xe2x80x94N(CH3)NH2, xe2x80x94NHCON NH2xe2x80x94NHCSNHNH2 or phenyl hydrazine], haloacetamide (e.g. iodoacetamide or bromoacetamide) xe2x80x94NCO, xe2x80x94NCS, xe2x80x94COR10, [where R10is a halogen atom such as a chlorine or bromine atom, or a N3, C1-6 alkoxy, e.g. methoxy, C6-12 aryloxy (e.g. nitrophenyloxy or dinitrophenyloxy) imidyloxy (e.g. succinimidyloxy) or imidazolyoxy group], imide, e.g. maleimide, a vinyl group of formula -Het1-C(Het2)=CH2 (where Het1 and Het 2, which may be the same or different, is each a nitrogen containing heterocyclic group, e.g. a pyridyl group or Het1 is a nitrogen containing heterocyclic group and Het2 is a hydrogen atom), for example a vinyl pyridyl group of formula 
especially 
or 
or a dione of formula 
(where R11 is a C1-4alkyl, e.g. methyl, group) .
In general, compounds of formula (1) in which the reactive functional groups are the same are preferred, although for some uses it may be preferable to have more than one type of reactive functional group. Particularly preferred functional groups are those capable of reacting with thiol groups. Groups of this type include imide (particularly maleimide), haloacetamide (particularly iodoacetamide), xe2x80x94SH, Het-Sxe2x80x94Sxe2x80x94, -Het1(Het2)=CH2 or 
groups. Imide, especially maleimide, groups are particularly useful.
The compounds of formula (1) may contain three or more reactive functional groups, depending on their intended use.
Useful compounds include those containing three or four reactive functional groups, particularly three of four thiol-reactive groups, e.g. three or four maleimide groups, although if desired five, six, seven or eight such groups may be present.
The reactive functional groups may be distributed in the groups R2 and R3 is any desired way. Thus, for example, each of R2 and R3 may contain 1, 2, 3 or more reactive functional groups (providing the total number in both is three or more). Alternatively, the reactive functional groups may be in one of R2 or R3 only.
The groups R2 and R3 in compounds of formula (1) form a template to which the reactive functional groups are attached and may be varied within any desired size and composition. Thus, one particularly preferred, but not limiting, group of compounds of the invention has the formula 1) wherein R1 is as defined above and R2, which may be the same or different is each an optionally substituted straight or branched C1-25 alkylene (e.g. C1-16 alkylene such as methylene, ethylene, propylene, butylene, pentylene, hexylene or heptylene), C2-23 alkenylene or C2-20 alkynylene chains, [optionally interrupted by one or more xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94 atoms, xe2x80x94N(R4)xe2x80x94 (where R4 is a hydrogen atom or a C1-6 alkyl group such as a methyl or ethyl group), xe2x80x94N(R4)COxe2x80x94, xe2x80x94CON(R4)xe2x80x94 C5-8 cycloalkylene (e.g. cyclopentylene or cyclohexylene), C6-12 arylene (e.g. phenylene or substituted phenylene) or C5-10 heteroarylene (e.g. furanyl, thienyl or pyridinyl groups)] containing one or more reactive functional groups such that the total number of reactive functional groups in R2 and R3 together is three or more.
Optional substituents present in the groups R2 and R3 include carboxyl (xe2x80x94CO2H) and esterified carboxyl (xe2x80x94CO2R) [where R is as defined above] and amino (xe2x80x94NH2) or substituted amino (NR6R7) [where R6 and R7, which may be the same or different, is each a hydrogen atom or a C1-6 alkyl group, or a group xe2x80x94COR8 where R8 is as defined for R2, providing that when one of R6 and R7 is a hydrogen atom, the other is not, and when one of R6 and R7 is a group xe2x80x94COR8, the other is a hydrogen atom].
It will be appreciated that when one, or both of R2 and R3 contains a substituent xe2x80x94NHCOR8 this allow for further reactive functional groups to be built into the compound of formula (1).
Particularly useful groups R2 or R3 may have a structure xe2x80x94(CH2)mNHCOCH(NHCOR8)(CH2)nNHCO(CH2)pZ (where m, n and p, which may be the same or different is each an integer 1, 2 3 or 4 and Z is a reactive functional group as defined above.
Particularly useful groups of compounds of the invention have the formula (6): 
or the formula (7): 
in particular the formula (8): 
or the formula (9): 
Another preferred cross-linking agent has the formula (10):
R9CH (R2)CONHCH(R2)CONHCH(R2)CONH2xe2x80x83xe2x80x83(10)
where R9 is xe2x80x94NH2 or a substituted amino group, e.g. a group xe2x80x94NHCOA, and A and R2 are as defined for compounds of formula (1). It will be appreciated that in compounds of this type each R2 group may be the same or, if desired, different to its neighbour.
The compounds of formulae (1) and (10) are of particular use for cross-linking biological materials, especially proteins, and in particular antibodies, providing the biological material(s) have one or more functional groups capable of reacting with the compound of formulae (1) or (10). The compounds are particularly useful for producing TFM and QFM compounds according to the invention.
The cross-linking reaction may be achieved using conventional processes, for example by mixing the starting materials, such as Fab fragments and the appropriate linker, in an aqueous solvent, e.g. at ambient temperature. The relative concentrations of the starting materials used will depend to a large extent on the compound of formula (1) or (10) and the number of reactive functional groups it contains, and the nature of the desired product, but generally the biological material(s), e.g. proteins such as an antibodies, e.g. a Fab fragment, will be present in excess concentration.
The compounds of formulae (1) and (10) may be prepared by a number of processes, for example as described in the examples appended hereto. In these processes reactive groups may need to be protected, when it is desired that they do not participate in a particular reaction. Conventional carboxylic acids may be esterified (for example to generate benzyl esters) and amino groups may be acylated (for example to generate benzyloxycarbonylamino groups). The protecting groups may be removed using conventional procedures, for example in the case of a benzyl ester by treatment with an acid, e.g. formic acid, and in the case of a benzyloxycarbonylamino group by treatment with a compound such as trimethylsilyl iodide.
Thus, for example, compounds of formula (1) wherein R1 is a group xe2x80x94COA or xe2x80x94COxe2x80x94SP-A may be prepared by reaction of a corresponding compound wherein R1 is a group xe2x80x94CO2H or an activated derivative thereof (for example a succinimide, e.g. obtained by reaction of the acid with N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide) with the group A or SP-A, optionally in the presence of a base, in a solvent such as an ether, e.g. a cyclic ether such as tetrahydrofuran. In this reaction the starting material A or Sp-A will require a group capable of reacting with the acid-activated derivative thereof. Such groups include, for example, amino and hydroxyl groups.
Compounds of formula (1) wherein R1 is a xe2x80x94CO2H group may be prepared by hydrolysis of the corresponding ester (xe2x80x94CO2R), using conventional procedures, for example by hydrolysis using an acid e.g. trifluoroacetic acid, in an inert solvent such as a halogenated hydrocarbon.
In general the compounds of formula (1) in which R1 is a xe2x80x94CO2R group may be prepared in a step-wise fashion from an esterified amino-acid starting material of formula (11):
ROOCCH(Y)NH2xe2x80x83xe2x80x83(11)
[where Y is a side chain containing a reactive group [e.g. an amino (xe2x80x94NH2) group] or a displaceable group (e.g. a halogen atom)] using a series of displacement or condensation reactions involving other intermediates with appropriate reactive groups using conventional procedures. The general synthetic principle may be illustrated by reference to the intermediates and examples described herein where the preparation of certain compounds according to the invention is illustrated using a known starting material. Other compounds according to the invention may be prepared using the same approach but with different starting materials and intermediates to introduce other types of groups R2 and R3 containing different reactive functional groups.
compounds of formula (10) may be prepared in analogous fashio using displacement and condensation reactions for example as illustrated in the intermediates and examples set out herein.
The performance of any suitable reactive functional group is always subject to the structural constraints placed upon it by the linker molecule itself. It has been found that increased linearity of the linkers facilitates the addition of a macrocycle and the chelation of an effector group.
Therefore, the invention comprises novel linkers which have a substantially linear backbone structure and are capable of accomodating a macrocycle group.
Particularly preferred are ligands of the formula: 
wherein R1 is as described above and Mal is a maleimide group.
Preferably, the TFM or QFM of the invention is a tri- or tetra-valent monospecific antigen-binding protein comprising three or f our Fab fragments bound to each other by a linker having attached thereto a macrocycle.
It will be understood that although the attachment of the macrocycle to the linkers of the invention is preferred, it is also possible to attach the macrocycle to one or more of the Fab fragments, incorporated into the multi-valent proteins of the invention. This approach is particularly preferred where the linker used does not facilitate the attachment of a macrocycle group.
Preferably, therefore, the TFM or QFM of the invention contains a radiolabel. The radiolabel is chelated by the macrocycle.
In a further preference the Fab fragment in each TFM or QFM compound according to the invention are bound to each other by a connecting structure linked to a thiol group on each Fab fragment.
Particularly preferred are tri- or tetra-valent protein constructs of the invention in which the connecting structure is one of the following linkers: 
Naturally-occurring Fabxe2x80x2 fragments have a number of thiol groups in the hinge region, typically two, four or even eleven. In an advantageous embodiment of the present invention, however, genetically modified Fabxe2x80x2 fragments are used which have only a single free thiol group in the hinge region. Construction by recombinant DNA technology of such Fabxe2x80x2 fragments, referred to as xcex4 cys Fabxe2x80x2 fragments, is described in our copending European patent application No. 0347433.
Decreasing the number of cysteine residues in the hinge region of a Fab-like fragment such as a Fabxe2x80x2 advantageously decreases the possibilities of incorrect interaction between the Fab-like molecule and the linker molecule.
Normally, purified Fabxe2x80x2 fragments produced by recombinant DNA technology are recovered with blocked hinge thiol groups. In this instance, Fabxe2x80x2 fragments are preferably partially reduced before assembly into TFM or QFM compounds of the invention.
Preferably, the Fabxe2x80x2 fragments are cross linked using a cross-linker of formula (1) or (10). Most preferably, the cross-linker is one of the structures depicted in the examples attached hereto. Most preferably, the linker is
The tri- or tetra-valent monospecific antigen binding proteins of the invention may be used for in vivo diagnosis or therapy.
Thus the invention also includes tri- or tetra-valent monospecific antigen-binding proteins according to the invention having attached thereto diagnostically or therapeutically functional effector molecules, atoms or other species. Any of the effector or reporter groups described above may be included.
The proteins of the invention are of use for In vivo diagnostic or therapeutic purposes. Thus, the invention also includes diagnostic or therapeutic compositions for in vivo use comprising an effective amount of a protein according to the invention in combination with a pharmaceutically acceptable diluent, excipient or carrier.
The composition may comprise other active ingredients.
The composition may take any suitable form for administration, and may, in particular, be in a form suitable for parenteral administration, e.g. by injection or infusion, for example by bolus injection or continuous infusion. Where the composition is for injection or infusion it may take the form of a suspension, solution or emulsion of the protein of the invention in an oily or aqueous vehicle and it may contain formulatory agents such as suspending stabilising and/or dispensing agents. Alternatively, the compositioin may be in a dry form, for reconstitution before use with an appropriate sterile liquid.
The dose at which the protein according to the invention may be administered will depend on whether the protein is being used for diagnosis or treatment, on the nature of the condition to be diagnosed or treated, on whether the protein is being used prophylactically or to treat an exisiting condition and on the particular Fab fragment and effector or reporter group selected. Dose will also be selected according to age and condition of the patient. Thus, for example, doses in the range 0.01 to 10 mg/Kg/day may be used. Advantageously, since the compounds according to the invention are cleared rapidly from the blood, multiple dosing regimes may be used.
Moreover, the invention includes methods of diagnosis or therapy comprising administering an effective amount of a protein of the invention to a human or animal subject.
Most preferably, the method of the invention is directed to the treatment or diagnosis of cancer.
The invention further comprises the use of a tri- or tetra-valent protein as described in the preceding aspects of the invention for the treatment of an ailment, preferably cancer. Furthermore, the invention comprises the use of a tri- or tetra-valent protein according to the invention in the manufacture of a composition for the treatment of the ailment, which is preferably cancer.
The present invention is now described, by way of example only, with reference to the accompanying drawings, in which: